Down‐regulation of wheat Rubisco activase isoforms expression by virus‐induced gene silencing

Abstract Rubisco activase (Rca) is an essential photosynthetic enzyme that removes inhibitors from the catalytic sites of the carboxylating enzyme Rubisco. In wheat, Rca is composed of one longer 46 kDa α‐isoform and two shorter 42 kDa β‐isoforms encoded by the genes TaRca1 and TaRca2. TaRca1 produces a single transcript from which a short 1β‐isoform is expressed, whereas two alternative transcripts are generated from TaRca2 directing expression of either a long 2α‐isoform or a short 2β‐isoform. The 2β isoform is similar but not identical to 1β. Here, virus‐induced gene silencing (VIGS) was used to silence the different TaRca transcripts. Abundance of the transcripts and the respective protein isoforms was then evaluated in the VIGS‐treated and control plants. Remarkably, treatment with the construct specifically targeting TaRca1 efficiently decreased expression not only of TaRca1 but also of the two alternative TaRca2 transcripts. Similarly, specific targeting of the TaRca2 transcript encoding a long isoform TaRca2α resulted in silencing of both TaRca2 alternative transcripts. The corresponding protein isoforms decreased in abundance. These findings indicate concomitant down‐regulation of TaRca1 and TaRca2 at both transcript and protein levels and may impact the feasibility of altering the relative abundance of Rca isoforms in wheat.

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Sincerely, Ana Fortes
- In this paper, the authors try to silence independently the three Rubisco activase isoforms in the hexaploid wheat species.VIGS was chosen to lead this approach, as this is an effective way of inducing silencing, although accompanied by some viral symptoms.The authors observe that their use of isoformspecific sequences on either the Rca1 or Rca2 genes to induce silencing leads to the reduction of the targeted transcript and corresponding protein accumulation, however the reduction is also observed on the untargeted transcript.They conclude that the two Rca genes are coregulated.The authors also examine the effects of reduced expression of Rca on Rubisco accumulation, but do not observe any effect.In my opinion, in its current state, the conclusions are not supported strongly enough by the experiments.The main conclusion states that Rca paralogs are coregulated because silencing one Rca isofom affects the expression of the second.This conclusion seems an overstatement as the results may be explained by other hypotheses related to the mechanism of VIGS silencing.Although VIGS silencing is widespread, it is usually not used to silence a specific member of a gene family.This is because controlling its effect on a gene family is difficult.First, the region used to convey the silencing may act in trans on the other paralogs because it displays too much homology to the other paralogs.This point is raised by the authors in the discussion, but according to them, this is not the case for the regions used in this work.Indeed the regions used to silence specifically Rca1 would seem divergent enough not to target Rca2.Similarly in the case of the Rca2 alpha-specific form, the region used is not present in the Rca1 gene.However, the region used to silence both isoforms (Rca2 alpha and beta) stemming from the Rca2 gene, shows several conserved blocks of more than 23 nt (at least 3) in both Rca1 and Rca2 paralogs.Silencing in trans could thus occur leading to downregulation of all Rca isoforms as observed here.Indeed, this construct is the most efficient one, resulting in transcript and protein reduction for all three isoforms.The second point that could be raised here is that VIGS can induce "transitive silencing" (Bleys A, Van Houdt H, Depicker A. Down-regulation of endogenes mediated by a transitive silencing signal.RNA.2006 Sep;12(9):1633-9.doi: 10.1261/rna.108106,for a review: Sanan-Mishra et al., Front.Plant Sci., https://doi.org/10.3389/fpls.2021.610283)).Transitive silencing could be completely compatible with the observations made here: silencing of Rca1 could induce secondary sRNA downstream of the targeted sequence, whose sequence would be homologous enough to induce the silencing of the Rca2 paralog.Curiously, this possibility is not discussed at all in the manuscript.To support the coregulation hypothesis raised by the authors, it would be necessary to be able to rule out whether the effect could not be due to transitive silencing, which could be experimentally assessed through the detection of these sRNA.Beside this major concern about the methodology used, additional points would need further examination: -line 40: correct "Rubisco binding protein" to "Ribulose 1,5 bisphosphate" -result section: Table 2: the primers used for qRT-PCR for Rca2b seem to be incorrectly described.The reverse primer is the one used for Rca2alpha, the forward primer could not be found within the provided sequence.Please clarify this point.It would be easier for the reader to have a visual localization of the primers used on the sequence provided in figure S1. -Figure S1: the 3' end sequence of Rca1betaD (bp 1281 to 1300) is curiously close to Rca2 beta A and B, while the Rca2beta D is close to the Rca1beta A and B. Please check whether this is correct or a problem in the alignment.Also, the VIGS targeted regions are indicated in the legend to be below the sequence.However this is not the case for rca1beta and rca2, where the red line is incorrectly placed.
-line 229: authors indicate that Rca1 beta level remains unchanged upon silencing with the Rca2alpha specific region, shown to induce Rca 2alpha and beta reduction (median around 60% of levels observed in the control.This is based on the statistical analysis of the Rca1beta transcript between the control and VIGS-Rca2alpha silenced plants, but may seem a bit too strong according to the figure, in which the Rca1beta median is around 80% compared to the control.-Discussion section; Line 272: please add BSMV::Rca2alpha showed a MILD decrease... -line 313: overstatement: in line with the reduction of transcript levels (instead of coregulation) -Rubisco abundance is measured by Ponceau stain.To be able to discuss Rubisco accumulation, this method seems very imprecise, and standard immunoblotting with commercially available Rubisco antibodies would seem more appropriate.
-lines 349-351: as mentioned in the general points, this conclusion is poorly supported by the data provided.
Minor points: -line 84: the authors claim that little is known concerning the regulation of Rubisco activase isoforms although they showed in a previous report that Rca1 is induced by heat.This could be mentioned here.
-line 128: reference to Yuan et al., 2011 is not found in the bibliography section.
-MM section, line 177 indicated that ten biological replicates have been collected while table S2 indicated 8.
Reviewer #2: • Please explain critically the potential application of the current findings at large scale in agricultural fields.This should be added in the conclusion section.
• Lack of use of articles in several places, for example, in L41 "the" unproductive binding, "the" active sites; in L107 "the" production.Please check the whole manuscript and do the necessary corrections.• L80-81: Use of "the more highly expressed" is not technically sound.• L84-85: There are some recent papers available on regulation of the Rubisco activase isoforms expression in wheat, especially under heat stress.
• Line 95: Please use either "2-3 weeks" or "two to three weeks" instead of "two-three weeks".